Cell-bound keratinolytic enzymes or trichophyton mentagrophytes and process therefor

ABSTRACT

Two cell-bound keratinases are produced by incubating T. mentagrophytes for several days in growth medium, at a pH of 7.8, containing horse hair, magnesium salt, thiamine, pyridoxine and inositol. Preferably, glucose is also included. These keratinases are extracted from the fungus mycelium after filtering off the growth media, partially purifying the enzymes by adsorbing them separately on diethylaminoethyl cellulose and carboxymethyl cellulose. The first enzyme which is eluted from the diethylaminoethyl cellulose and purified by molecular sieves has a molecular weight of 440,000. The second enzyme which is eluted from the carboxymethyl cellulose and purified by molecular sieves has a molecular weight of 20,300. Physicochemical properties as well as their keratinolytic activities have been determined.

United States Patent [191 Blank et a1.

[ Feb. 19, 1974 [75] Inventors: Fritz Blank, Merion; Ruey J. Yu,

' Philadelphia, both of Pa.

[52] US. Cl 195/66 R, 195/65, 195/62 [51] Int. Cl C07g 7/02 [58] Field of Search 195/62, 65, 66 R, 4, 5, 6

[56] References Cited UNITED STATES PATENTS 3/1965 Kita et a1. 195/5 OTHER PUBLICATIONS Yu et al., The Journal of Investigative Dermatology Vol. 56, No. 1, Jan. 1971, pages 27-32. Chemical Abstracts Vol. 72, 39206h (1970).

Britne E inekyw hawl? Attorney, Agent, or Firm -Paul & Paul [57] ABSTRACT Two cell-bound keratinases are produced by incubating T. mentagrophytes for several days in growth medium, at a pH of 7.8, containing horse hair, magnesium salt, thiamine, pyridoxine and inositol. Preferably, glucose is also included. These keratinases are extracted from the fungus mycelium after filtering off the growth media, partially purifying the enzymes by adsorbing them separately on diethylaminoethyl cellulose and carboxymethyl cellulose. The first enzyme which is eluted from the diethylaminoethyl cellulose and purified by molecular sieves has a molecular weight of 440,000. The second enzyme which is eluted from the carboxymethyl cellulose and purified by molecular sieves has a molecular weight of 20,300. Physicochemical properties as well as their keratinolytic activities have been determined.

7 Claims, No Drawings CELL-BOUND KERATINOLYTIC ENZYMES OR TRICHOPHYTON MENTAGROPHYTES AND PROCESS THEREFOR INTRODUCTION This invention pertains to a method for producing two cell-bound keratinolytic enzymes and to the product of that process.

BACKGROUND OF THE INVENTION Keratin is a sulfur containing protein which forms the chemical basis of such relatively insoluble and undigestible epidermal material as hair, nail, callus, horn, feather and stratum comeum. An enzyme capable of digesting or solubilizing keratin may have both dermatological and industrial uses. Dissolving keratinous waste material or the removal of hair in the rendering of animal hides are typical of the possible industrial uses of such an enzyme. Dermatologically, those keratinolytic enzymes would find utility, for example, in the removal of calluses or the excessive keratin found in certain skin conditions, such as psoriasis. Further potential use might include immunization of human beings and animals with such enzymes for the protection against fungus infection.

It is the general object of the present invention to provide such enzymes and methods for the production thereof.

BRIEF DESCRIPTION OF THE INVENTION Two active cell-bound keratinases are produced by incubating Trichophyton mentagrophytes in an aqueous growth medium having a pH of 7.8 and comprising horse hair, a magnesium salt, thiamine, pyridoxine and inositol. Preferably, glucose is also included to initiate the growth. Two cell-bound keratinases produced by the fungus are best isolated by first filtering off the incubated medium, then homogenizing the mycelium in phosphate buffer containing sodium chloride and successively contacting the buffer solution after dialysis with diethylaminoethyl cellulose and carboxymethyl cellulose. The first enzyme which is adsorbed on diethylaminoethyl cellulose is eluted with a buffered sodium chloride solution, dialyzed and lyophilized. The crude enzyme thus isolated is redissolved in buffered ionic solution and purified by molecular sieves such as Sephadex G-l00. The product which has a molecular weight of 440,000 is highly keratinolytic, as shown by its ability to solubilize human callus tissue, calf hoof and guinea pig hair. The second enzyme which is adsorbed on carboxymethylcellulose is eluted with a buffered sodium chloride solution, dialyzed and lyophilized. The crude enzyme thus isolated is redissolved in buffered ionic solution and purified by molecular sieves such as Sephadex G-lOO. The product which has a molecular weight of 20,300 is highly keratinolytic, as shownby its ability to solubilize human callus tissue, calf hoof, guinea pig hair and horse hair.

DETAILED DESCRIPTION OF THE INVENTION The present invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. It may be better understood, however, together with the further objects and advantages thereof, by reference to the following detailed descriptron.

Trichophyton mentagrophytes var. granulosum (T. granulosum Sabouraud, 1909) is a known dermatophyte. Dermatophytes are parasitic fungi known to cause diseases such as ringworm in man and animals. The main pathogenic feature of dermatophytes is their ability to invade and thrive in the keratinized layers of skin, hair and nails. While this physiological property of dermatophytes has been known for some time, efforts to isolate and characterize derrnatophytic enzyme with specific keratinolytic activity have met with only one example. The same present inventors isolated an extracellular keratinolytic enzyme from the culture medium of T. mentagrophytes in 1968. This extracellular enzyme was studied in detail and was well characterized. The present invention is particularly directed to the isolation and characterization of two cell-bound enzymes of the same species, which are nevertheless new and different from any previously known enzyme. Production Two cell bound enzymes are produced according to the present invention by inoculating a culture medium with T. mentagrophytes. Generally, the culture medium is composed of the following components in grams per liter of distilled water:

Horse hair 2.5

Glucose 0.9

Thiamine hydrochloride 0.01

Pyridoxine hydrochloride 0.01

Inositol 0.05

Na HPO 7H O (6.87 grams) and KH PO (0.333

grams) are added to produce a buffered pH of 7.8. Horse hair may be varied from about 1 to 5 grams. The magnesium concentration, about 5 millimole in the foregoing composition, may be varied within the range 1-10 millimoles. The buffered pH may be varied from 7 to 9.

After sterilization at 121 C and 15 psi for 20 minutes, the culture medium is inoculated with T. mentagrophytes and is kept in the dark for 5 days at 28 C. The culture medium is then shaken for 7 days and filtered. The mycelium is washed first with cold water then with cold acetone to remove lipids.

Isolation and Purification Crude cell-bound enzymes are extracted from the mycelium by repeated blendoring in 0.01 M phosphate buffer, pH 7.8 containing 0.5 M NaCl and by dialyzing. Two enzymes in the dialyzed extract are adsorbed separately on diethylaminoethyl cellulose and carboxymethyl cellulose. The first enzyme which is adsorbed on diethylaminoethyl cellulose is eluted with 0.5 M NaCl in 0.01 M phosphate buffer, pH 7.5, dialyzed and lyophilized. Purification of this enzyme is accomplished by dissolving the lyophilized crude enzyme in 0.01 M phosphate buffer, pH 7.5, followed by gel filtration in a chromatographic separation column with a gel filtration agent such as Sephadex G-200 or G-lOO molecular sieve. The first enzyme has a molecular weight of 440,000 as determined by the method of gel filtration on Sephadex G-200. The second enz yme'which is adsorbed on carboxymethyl cellulose is eluted with 0.5 M NaCl in 0.01 M phosphate buffer, pH 7.8, dialyzed and lyophilized. Purification of the second enzyme is accomplished by dissolving the lyophilized preparation in 0.01 M phosphate buffer, pH 7.5, followed by gel filtration in a chromatographic separation column with a gel filtration agent such as Sephadex G-l molecular sieve. The second enzyme has a molecular weight of 20,300 as determined by the method of gel filtration on Sephadex G-100. Keratinolytic Activity The keratinolytic activities of both enzymes produced by the foregoing process and the enhancement thereof as they are purified are best illustrated by the following table in which the fluid volume, total keratinolytic activity and specific keratinolytic activity (keratinolytic activity per milligram enzyme in solution) are given for the fluid at each stage of the purification process. The keratinolytic activity is given in units based on spectrophotometric analysis of reaction fluids of guinea pig hair as digested by enzyme material.

Keratinolytic Activity of Two Cell-Bound Keratinases 'Histidine 7.57 14.65 lsoleucine 2.27 3.62 Leucine 4.54 3.62 Lysine l 1.64 4.84 Methionine 0.51 0.82 Phenylalanine 3 .43 3.64 Proline 6.78 5.72

, Serine 6.91 5.80 Threonine 7.01 7.23 Tryptophan 3.53 2.25 Tyrosine 5.64 3.08 Valine 3.24 5.17

Total 99.90 100.00

The carbohydrate compositions of these two keratinases (polysaccharide portion) in grams per 100 grams, are as follows:

Procedure Volume Total Specific (milliliters) Activity Activity (x KU) g) 1. Mycelial extract 1,102 3.05 19.9

First Keratinase 2A. DEAE-cellulose 215 0.46 23.4 3A. Sephadex 0-100 12 0.29 36.4

Second Keratinase 2B. CM-cellulose 329 1.43 29.1 38. Sephadex G-100 53 0.91 39.4

In other tests of the keratinolytic properties of the First keratinase second keratinase two keratinases of the present invention, the keratin Mannose 81.0 66.7 content of human callus material in a reaction solution Galacmse 150 2&7 at 37 C and a pH of 7.0 wasfound to be 18 percent (by Glucose 4.0 6.7 weight) digested by the first keratinase and 36 percent Tom] 100 0 (by weight) digested by the second keratinase after 16 hours. Physicochemical Properties The physicochemical characteristics of the two keratinases of the present invention have been determined. The isoelectric points of the first and second keratinases are pH 4.40 and pH 9.85, respectively. Their elemental analyses, by weightare:

First keratinase Second keratinase Carbon 48.53 40.93

Hydrogen 7.67 6.54 Oxygen 38.39 46.09 Nitrogen 3 .05 5.01 Sulfur None None Ash 2.38 1.67

The amino acid compos itions of these two keratinases (protein portion) in grams per 100 grams, are as Subunit Structure After these two pure keratinases were treated with mercaptoethanol and sodium dodecylsulfate the first enzyme was split into 9 different subunits with molecular weights of 6,100, 16,000, 18,200, 26,500, 30,000, 31,500, 41,500, 52,000 and 62,500,. whereas thesecond enzyme was split into one identical subunit with molecular weight of 6,100. pH Optimum The two pure keratinases showed maximal enzymatic activity between pH 6.0 8.0 when guinea pig hair was used as substrate.

Optimum Temperature The two pure keratinases showed maximal enzymatic activity at about 46 C when guinea pig hair was used as substrate.

Optimum Ionic Strength Digestion of guinea pig hair by the two keratinases in sodium chloride solutions of various concentrations was maximal at 0.05 M Nacl in buffer, pH 7.0.

Effect of Metal Ions and EDTA Effect of various metal ions and EDTA at 10 mM concentration on keratinolytic activity (percent) of the two keratinases is best illustrated by the following data.

First keratinase Second keratinase Control 100 100 Ba 1 10 102 Ca 30 Cd 0 0 First keratinase Second keratinase Cu 0 M [00 l 10 Mn 53 45 93 42 Ag 0 0 0 0 g2 13 56 Fe 0 0 S 0 0 Al 9 0 EDTA l l4 AMIDES, PEPTIDES OR PROTEINS HYDROLYZED BY TWO KERATINASES OF THE PRESENT INVENTION L Ser-B-Naph-NH,

L Thr-B-Naph-NH L-Ala-B-Naph-NH Gly-B-Naph-NH -His-[3-Naph-NH 4. 5. 6. 7. 8. L Ile-B-Naph-NH HBr 10. ll. l2. l3. l4.

L-Met-B-Naph-NH L-Orn-B-Naph-NH L-Phe-B-Naph-NH L-Val-B-Naph-NI-I N-CBZ-a-Glu-L-Tyr 15. N-CBZ-Gly-L-Phe-NI-I l6. N-CBZ-L-Val-L-Tyr-OCH N-CBZ-L-Val-L-Phe-OCH N-CBZ-L-Val-L-Leu-OCI-I N-CBZ-Gly-L-Leu Gly-L-Phe Gly-DL-NorLeu a-GlyL-LysHCl Gly-L-Leu Gly-L-Tyr Gly-DL-Val L-Tyr-L-Leu N-CBZ-L-Leu-L-AlaOCH L-Leu-L-Tyr N-CBZ-L-Pro-L-Leu-Gly-NH Gly-L-Leu-L-Tyr L-Leu-L-Leu L-Leu-L-Met DL-Leu-Gly L-Tyr-Gly L-Phe-Gly L-Val-Gly L-Leu-L-Phe DL-Ala-Gly-Gly Poly-L-Lys I-ICl N-CBZ-Gly-L-Pro-L-Leu-Gly-L-Pro Bovine albumin Elastin powder 44. Bovine collagen 45. Bovine fibrinogen 46. Bovine fibrin 47. Bovine insulin 48. Bovine hemoglobin AMIDES, PEPTIDES OR POLYPEPTIDES NOT HYDROLYZED BY TWO KERATINASES OF THE PRESENT INVENTION L-Pro-B-Naph-NI-I N-Tosyl-L-Arg-B-Naph-NH L-Asn-,B-Naph-NH L-a-Asp-B-Naph-NH L-B-Asp-B-Naph-NI-I L-Hyp-B-Naph-NH L-Try-B-Naph-NH L-Tyr-B-Naph-NI-b N-BZ-L-Tyr-Gly-NH DL-Ala-DL-Ser N-CBZ-L-Ala-LSer-OCH N-CBZ-L-AlaI-Val-OCH; N-CBZ-L-Met-Gly-OCH Gly-L-Glu N-CBZ-L-Val-L-Ala-OCH N-CBZ-L-Tyr-Gly-NH N-BZ-Gly-Gly Gly-L-Ser Gly-L-Pro Gly-Gly N-CBZ-L-Ala-Gly-Gly L-Ser-Gly B-Ala-DL-Leu N-CBZ-L-Leu-L-Leu-NH Poly-L-Gly Poly-L-Leu Poly-L-Phe Poly-L-Glu In addition, Gly-L-Pro-Gly-Gly and L-y-Glu-B-Naph-NH are hydrolyzed by the first keratinase but not hydrolyzed by the second keratinase of the present invention.

We claim:

1. A process for making keratinolytic enzymes including:

a. preparing an aqueous growth medium consisting of a l-lO millimole solution of a magnesium salt having a pH of 7-9 and further including (per liter solution):

1-5 grarns horse hair .9 gram glucose .01 gram thiamine hydrochloride .01 gram pyridoxine hydrochloride .05 gram inositol b. inoculating said growth medium with Trichophyton mentagrophytes var. granulosum,

0. allowing said T.mentagr0phytes to incubate in said medium for several days,

d. filtering said inoculated, incubated medium to separate the liquid fraction from the mycelium thereof,

e. washing the mycelium to remove lipids therefrom,

f. repeatedly contacting and mixing said washed mycelium with a NaCl phosphate buffered solution having a pH of 7.8 and dialyzing said solution mixture, fon'ning a dialyzed extract,

3. A process, as recited in claim 1, wherein said enzyme adsorbent is carboxymethyl cellulose, said enzyme is eluted therefrom with a NaCl solution with a phosphate buffer having a pH of 7.8, dialyzed,*lyophilized and purified by dissolution in a phosphate buffered pH 7.5 solution and gel filtration.

4. A process, as recited in claim 1, wherein said mycelium is washed with water and acetone.

5. A process, as recited in claim 1, wherein said washed mycelium is repeatedly contacted and mixed with a 0.5 m NaCl, 0.01 M phosphate buffer solution.

6. A process, as recited in claim 2, wherein adsorbed enzyme is eluted from said diethylaminoethyl cellulose with a 0.5 M NaCl, 0.01 M'phosphate buffer'solution and said gel filtration is effected on molecular sieves.

7. A process, as recited in claim 3, wherein adsorbed enzyme is eluted from said carboxymethyl cellulose with a 0.5 M NaCl, 0.01 M phosphate buffer, pH 7.8

and said gel filtration is effected on molecular sieves.

UNITED-STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 vI Dated February 19, 1974 Fritz Blank and Ruey J. Yu Inventor(s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

35 U.S.C. 254 v Page 1, The title "CELL-BOUND KERATINOLYTIC ENZYMES OR TRICHOPHYTON MENTAGROPHYTES AND PROCESS THEREFOR" Should read -CELLBOUND KERA'I'INOLYTIC ENZYMES OF TRICHOPHYTON MENTAGROPHYTES AND PROCESS THEREFOR-. 5

Column 1, lines 1-3, "CELL-BOUND KERATINOLYTIC ENZYMES OR TRICHOPHYTON MENTAGROPHYTES AND PROCESS THEREFOR" should read --CELL-BOUND KERATINOLYTIC ENZYMES OF TRICHOPHYTON MENTAGROPHYTES AND PROCESS THEREFOR.

Column 4, line 58 "Nacl" should read -NaCl-.

Column 8, Claim 5, "0.5 m" should read O.5 M--.

Signe end e le this 1 th day of September 1974.

(SEAL) Attest:

McCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner of Patents FORM Po-1030 (10-69) USCOMM-DC s ws-wa 

2. A process, as recited in claim 1, wherein said enzyme adsorbent is diethylaminoethyl cellulose, said adsorbed enzyme is eluted therefrom with a NaCl solution buffered to pH 7.5 with phosphate buffer, said solution containing said enzyme is dialyzed and lyophilized and said lyophilized enzyme is purified by dissolution in a phosphate buffered pH 7.5 solution and gel filtration.
 3. A process, as recited in claim 1, wherein said enzyme adsorbent is carboxymethyl cellulose, said enzyme is eluted therefrom with a NaCl solution with a phosphate buffer having a pH of 7.8, dialyzed, lyophilized and purified by dissolution in a phosphate buffered pH 7.5 solution and gel filtration.
 4. A process, as recited in claim 1, wherein said mycelium is washed with water and acetone.
 5. A process, as recited in claim 1, wherein said washed mycelium is repeatedly contacted and mixed with a 0.5 m NaCl, 0.01 M phosphate buffer solution.
 6. A process, as recited in claim 2, wherein adsorbed enzyme is eluted from said diethylaminoethyl cellulose with a 0.5 M NaCl, 0.01 M phosphate buffer solution and said gel filtration is effected on molecular sieves.
 7. A process, as recited in claim 3, wherein adsorbed enzyme is eluted from said carboxymethyl cellulose with a 0.5 M NaCl, 0.01 M phosphate buffer, pH 7.8 and said gel filtration is effected on molecular sieves. 